Small molecule modulators of prb inactivation

ABSTRACT

The present invention provides a small molecule treatment of diseases/conditions caused by a virus carrying a viral oncoprotein. In one embodiment, the virus which carries the viral oncoprotein is HPV. The small molecule useful herein includes thiadiazolin-3,5-dione compounds having an optionally substituted aryl group bound to one nitrogen atom of said thiadiazolin-3,5-dione compound. The small molecules may also be administered with a compound which inhibits binding of HPV E6 to p53. In one embodiment, the thiadiazolin-3,5-dione compound has formula (I), or a pharmaceutically acceptable salt, prodrug, solvate, or metabolite thereof, wherein R 1  and R 2  are defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of pending US patentapplication Ser. No. 14/356,979, filed May 8, 2014, which is a 371 ofInternational patent application No. PCT/US2012/063683, filed Nov. 6,2012, (now expired) which claims the benefit of priority of U.S.provisional patent application No. 61/558,686, filed on Nov. 11, 2011.The disclosures of these parent applications are incorporated byreference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.CA094165 and GM071339 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

INCORPORATION-BY REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeledWST131APCT_ST25.txt”, was created on 10 Oct. 2012, and is 16 KB in size.

BACKGROUND

HPV has received considerable attention due to its role in human cancer.In particular, HPV is known to be the causative agent of a number ofepithelial cancers, most notably cervical cancer, a leading cause ofdeath for women worldwide. HPV is associated with more than 95% of allcervical cancers, the leading cause of cancer deaths of woman indeveloping countries due to high HPV infection rates and lack ofcomprehensive cervical Pap smear testing of susceptible women.

HPV infection has also been implicated to have a causative role in about20% of head and neck cancers, the majority of anal and vaginal cancers,and about 50% and 35% of vulvar and penile cancers, respectively. Thereare over 200 HPV genotypes known, and they fall under two general forms:low-risk and high-risk, which cause benign and malignant lesions,respectively. Two prophylactic vaccines are currently available,Gardasil™ and Cervarix® vaccines, which help prevent against infectionby the low risk HPV types 6 and 11 and high risk HPV types 16 and 18.While these vaccines target HPV types that cause more than 90% ofgenital warts and cervical cancer, they have no therapeutic utility,i.e., they cannot treat existing infection. Furthermore, theeffectiveness and longevity of these vaccines will not be known fordecades, further warranting a need for therapeutics.

What is needed in the art are treatment options for patients infectedwith oncoviruses, such as HPV.

SUMMARY OF THE INVENTION

In one aspect, composition (A) for treating a HPV mediated disease isprovided and contains a (i) a thiadiazolin-3,5-dione compound which hasan optionally substituted aryl group bound to one nitrogen atom of saidthiadiazolin-3,5-dione compound; and (ii) a compound which inhibitsbinding of HPV E6 to p53. In one embodiment, the thiadiazolin-3,5-dionecompound has formula (I), or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof, wherein R¹ and R² are definedherein.

In another aspect, methods for preventing disruption of pRb/E2Fcomplexes are provided and include administering, to a patient in needthereof, a compound of formula (I) or composition (A).

In a further aspect, methods for preventing interaction between pRb anda viral oncoprotein are provided and include administering, to a patientin need thereof, a compound of formula (I) or composition (A).

In yet another aspect, methods for preventing or a disease caused by avirus carrying a viral oncoprotein containing a LxCxE motif are providedand include administering, to a patient in need thereof, a compound offormula (I) or composition (A).

In still a further aspect, methods for preventing or treating neoplasticdisease are provided and include administering, to a patient in needthereof, a compound of formula (I) or composition (A).

In another aspect, a method for preventing HPV-E7 mediated E2Fdisplacement from pRb is provided and includes administering a compoundof formula (I) or composition (A) to a patient in need thereof.

In yet another aspect, a method for disrupting pRb/HPV-E7 complexes isprovided and includes administering a compound of formula (I) orcomposition (A) to a patient in need thereof.

In still a further aspect, a method for preventing or treating genitalwarts is provided and includes administering a compound of formula (I)or composition (A) to a patient in need thereof.

In another aspect, a method for preventing or treating neoplasticdisease caused by HPV, adenovirus, or SV40 is provided and includesadministering a compound of formula (I) or a composition (A) to apatient in need thereof.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a flowchart summary of the process for theidentification of compounds 1-7.

FIGS. 2A and 2B as described below provide data forthiadiazolin-3,5-dione compounds 1-7. GraphPad® software (Prism) wasused for IC₅₀ determination and their corresponding errors. To calculatethe IC₅₀ values, three independent dose-response curves were fit toone-site (Hill slope=1) sigmoidal-dose-response curves. The error barswere obtained from the standard errors generated by GraphPad® software.FIG. 2A includes IC₅₀ curves for compounds 1-7 and is a plot ofconcentration (log [compound] (μM)) vs. percentage (%) of E2F remainingbound to pRb. IC₅₀ curves were generated using the ELISA-based assaydescribed in Example 2 and correspond to the following:

-   -   circles (): compound 2    -   dark, small squares (▪): compound 3    -   small triangles (▴): compound 6    -   inverted triangles (▾): compound 1    -   diamonds (♦): compound 4    -   light, large squares (▪): compound 5    -   large, triangles (▴): compound 7

FIG. 2B illustrates the cellular toxicity for compounds 1-7, asdiscussed in Example 2 (iv), with the results represented by a bargraph. The graph provides the percent growth of four different cervicalcancer cells lines, i.e., SiHa, TC-1, HeLa and C-33A and onenon-cervical cancer cell line, HCT116, in the presence of compounds 1-7and staurosporine, which is a positive control due to its expectedtoxicity in all cells. In each compound result group, the vertical baron the farthest left represents the percentage growth of the SiHa cells,the second left-most vertical bar for each compound represents thepercentage growth of the TC-1 cells, the middle vertical bar for eachcompound represents the percentage growth of the HeLa cells, the fourthleft-most vertical bar for each compound represents the percentagegrowth of the C-33A cells, and the fifth left-most vertical bar for eachcompound represents the percentage growth of the HCT116 cells.

FIGS. 3A and 3B as described below provide data illustrating disruptionof HPV-E7/pRb complexes using compounds 1-7. GraphPad®, software (Prism)was used for IC₅₀ determination and their corresponding errors. Tocalculate the IC₅₀ values, two independent dose-response curves were fitto one-site (Hill slope=1) sigmoidal-dose-response curves. The errorbars were obtained from the standard errors generated by GraphPad®software. FIG. 3A includes IC₅₀ curves for compounds 1-7 and the abilityof compounds 1-7 to disrupt HPV-E7/pRb complexes. IC₅₀ curves weregenerated using the ELISA-based assay described in Example 2 andcorrespond to the following:

-   -   circles (): compound 2    -   dark, small squares (▪): compound 3    -   small triangles (▴): compound 6    -   light, inverted triangles (▾): compound 1    -   diamonds (♦): compound 4    -   light, large squares (▪): compound 5    -   large, inverted triangles (▾): compound 7

FIG. 3B is a western blot and illustrates the effect of compound 2 onHPV-E7/pRb pull-down. Concentrations of compound 2 (0, 0.01, 0.1, 1, 10,and 100 μM) were independently added and the amount of GST-E7_(FL)remaining bound to pRb was probed by using an anti-GST antibody (bottompanel). The top panel shows the loading control of His-pRb_(ABC) in eachlane. The values above each panel provide the quantitative percentage ofpRb and GST-16E7_(FL) protein detected following pull-down at eachinhibitor concentration.

FIGS. 4A-4D as described below illustrate the effect ofthiadiazolin-3,5-dione compound 2 against viral oncoproteins E7 fromhigh HPV (type 16), 1AE7 from low risk HPV (type 1A), and E1a fromadenovirus. GraphPad® software (Prism) was used for IC₅₀ determinationand their corresponding errors. To calculate the IC₅₀ values, threeindependent dose-response curves were fit to one-site (Hill slope=1)sigmoidal-dose-response curves. The error bars were obtained from thestandard errors generated by GraphPad® software. FIG. 4A is a plot of %E2F vs. concentration (log [compound] μM) and illustrates E2F disruptionE2F/pRb complexes by LxCxE containing viral oncoproteins 16E7 (circles()), 1AE7 (squares ▪)), or E1A (triangles (▴)) in the presence ofcompound 2 by adding ten-fold dilutions of compound 2 toGST-pRb_(ABC)/6×His-HPV1AE7_(CR2-3) orGST-pRb_(ABC)/6×His-Ad5E1A_(CR2-3). FIG. 4B is a plot of % E7 vs. (log[compound] μM) and illustrates the disruption of complexes between pRband LxCxE containing viral oncoproteins 16E7 (circles ()), 1AE7(squares ▪)), or E1A (triangles (▴)) in the presence of compound 2. FIG.4C shows the binding of thiadiazolin-3,5-dione compound 2 to pRb as aplot of the molar ratio of compound 2 vs. integrated heats (kcal/mole)of compound 2 as measured by isothermal titration calorimetry.Integrated heats show the binding of compound 2 directly to pRb. Thecurve fit to a 1:1 binding model, with a K_(D) of 165 nM. FIG. 4D showsthe ability of HPV-E7 to disrupt complexes of pRb and compound 2 usingincreasing concentrations (0.025, 0.25, 05, and 5 μM) of compound 2. Theplot is a measure of concentration (log [E7] (μM)) vs. % E7. Five-folddilutions of E7 were added to pRb/compound 2 complexes and the amount ofE7 that was able to bind to pRb was determined.

FIG. 5A-5D as described below illustrate the reversibility of compound 3in binding to pRb as measured by isothermal titration calorimetry. FIGS.5A-5B show the incremental heat effects of 10 μL titrations of 750 μM ofcompound 3 into pRb and buffer, respectively, and plot the heatgenerated (μcal/sec) vs. time (min). FIG. 5C shows the incremental heateffects of 8 μL titrations of compound 3 into pRb solutions afterpRb/compound complexes were dialyzed overnight to remove the unboundcompound and is a plot of the heat generated (μcal/sec) vs. time (min).This binding curve provides a dissociation constant and stoichiometry,indicating that compound 3 interacted with pRb in a reversible fashion.FIG. 5D represents the binding curves for 6×His-HPV16-E7_(CR2-3)mediated displacement of E2F_(MB-TA) from GST-pRb_(ABC) in the presenceof varying concentrations of inhibitor, above and below the dissociationconstant of pRb for inhibitor. This data shows a dependence on theconcentration of inhibitor used, where increasing inhibitorconcentration is correlated with a rightward shift (higher apparentvalue) in the IC₅₀ values for HPV-E7 mediated displacement of pRb/E2Fcomplex. This data suggests that inhibitor and HPV16-E7 bindcompetitively to pRb. Taking this result together with the observationthat these inhibitors are also able to disrupt pRb complexes withHPV1A-E7 and AD5-E1a (FIG. 5A) suggests that these inhibitors also bindpRb competitively with other LxCxE containing oncoproteins.

DETAILED DESCRIPTION OF THE INVENTION

In recognizing the need in the art for therapies for treating diseasescaused by certain oncoviruses, the inventors identified a family ofsmall molecules based on a thiadiazolin-3,5-dione backbone. Thesethiadiazolin-3,5-dione compounds unexpectedly inhibited the ability ofviral oncoproteins to disrupt pRb/E2F complexes by undesirablydisplacing E2F and thereby inactivating the function of the pRbtranscription factor. This finding is integral in the treatment ofcertain cancers where there are no known small molecule drug therapies.

As used herein, retinoblastoma transcription factor (referred to as“pRb” herein) is a protein that regulates cell cycle, apoptosis anddifferentiation through its direct binding to and inhibition of the E2Ffamily of transcription factors. pRb becomes phosphorylated bycyclin/cyclin dependent kinases (cdks), which then signals pRb torelease E2F proteins to transcribe genes necessary for the progressioninto the S-phase of the cell cycle, as well as for DNA replication. TheA and B cyclin fold domains of pRb form a “pocket” region of theprotein, which forms a groove that makes high affinity contacts to thetransactivation domain of E2F. While the A/B pocket of pRb is importantfor its biological activity, the C-terminal domain is important for theformation of physiological pRb-E2F complexes. The C-terminal domain ofpRb makes contact with the marked-box region of E2F, although with alower affinity. This domain of pRb is also subject to cell-cycledependent posttranslational modifications, such as phosphorylation andacetylation, as well as the recruitment of cyclins/cdks.

pRb is also a target for inactivation by viral oncoproteins, includingthose specified below. Viral oncoproteins bind to hypophosphorylatedpRb, disrupting pRb/E2F complexes and thereby leading to dysregulatedentry into S-phase of the cell cycle and neoplasia. Each viraloncoprotein that inhibits pRb function employs a conserved LxCxE forhigh affinity pRb binding. The LxCxE motif from viral oncoproteinscontributes to disruption of the pRb/E2F complexes by binding to the pRbB domain. The C-terminal domain of pRb is the target of other regions ofthe viral oncoproteins. Each oncoprotein uses different protein regionsto displace pRb/E2F complexes through distinct mechanisms.

The inventors found that thiadiazolin-3,5-dione compounds bind directlyand competitively to the LxCxE binding site of pRb with dissociationconstants in the mid-high nanomolar range. The thiadiazolin-3,5-dionecompounds are also competitive for pRb binding to other viraloncoproteins containing an LxCxE motif. Therefore, in one embodiment,these thiadiazolin-3,5-dione are inhibitors. In yet another embodiment,the thiadiazolin-3,5-dione compounds prevent disruption of pRb/E2Fcomplexes. In a further embodiment, the thiadiazolin-3,5-dione compoundsprevent interactions between pRb and a viral oncoprotein.

These thiadiazolin-3,5-dione compounds are the first class of smallmolecules that competitively inhibit the interaction of LxCxEmotif-containing viral oncoproteins with pRb. The identification ofthese thiadiazolin-3,5-dione compounds is an important finding giventhat there are no known inhibitors that specifically block theinteraction of pRb with viral oncoproteins. In one embodiment, thethiadiazolin-3,5-dione compounds bind to pRb. In another embodiment, thethiadiazolin-3,5-dione compounds bind to the LxCxE binding site of pRb.In a further embodiment, the thiadiazolin-3,5-dione compounds preventone of the main transforming abilities of these oncoproteins, i.e., thepremature disruption of the inhibitory pRb/E2F complex. In still anotherembodiment, the thiadiazolin-3,5-dione compounds reduce or prevent pRbdegradation in HPV containing cells.

Of significance, the inventors found that these thiadiazolin-3,5-dionecompounds are highly selective. In one embodiment, thethiadiazolin-3,5-dione compounds exhibit selective cytotoxicity in HPVpositive cells.

The terms “patient” and “subject” are used interchangeably and refer toa mammal, preferably a human, who is infected with an oncogenic virusthat disrupts or inactivates normal pRb binding, such as HPV. Thepatient may be an adult or child. A “patient” or “subject” may alsoinclude a veterinary or farm animal, a domestic animal or pet, andanimals normally used for clinical research. In one embodiment, thepatient or subject is a human.

The terms “oncogenic virus” and “oncovirus”, which are usedinterchangeably, describe viruses that cause cancer. In one embodiment,the oncovirus causes or mediates malignant transformation of cells,inducing a neoplasia in a patient. In one embodiment, the oncovirus is apapovavirus such as human papilloma virus (HPV). In another embodiment,the oncovirus is a Herpes virus such as Kaposi's sarcoma-associatedherpes virus (KSHV or HHV-8) or Epstein-Barr virus (EBV). In a furtherembodiment, the oncovirus is a hepatitis virus, such as Hepatitis Bvirus (HBV) and Hepatitis C virus (HCV). In yet another embodiment, theoncogenic virus is an Adenovirus. In still a further embodiment, theoncovirus is a Poxvirus. In another embodiment, the oncovirus is aRetrovirus. In yet a further embodiment, the oncovirus is a Human T-cellLeukemia Virus. In another embodiment, the oncovirus is a polyoma virussuch as Merkel cell polyoma virus. In another embodiment, the oncovirusis simian virus 40 (SV40).

The term “viral oncoprotein” describes a viral protein that is involvedin the regulation or synthesis of proteins linked to tumorigenic cellgrowth. In a further embodiment, the viral oncoprotein targets, disruptsor inactivates pRb. In a further embodiment, the viral oncoproteincontains a LxCxE motif. In a further embodiment, the viral oncoproteinis E1a from adenovirus. In a further embodiment, the viral oncoproteinis E7 from high HPV. In a further embodiment, the viral oncoprotein is1AE7 from low risk HPV. In another embodiment, the viral oncoprotein isT-antigen from simian virus 40 (SV40).

The thiadiazolin-3,5-dione compounds, therefore, are useful in thetreatment or prevention of a variety of conditions/diseases. In oneembodiment, the thiadiazolin-3,5-dione compounds are useful in methodsfor preventing or treating a disease caused by an oncovirus containingan oncoprotein that targets, disrupts or inactivates pRb. In anotherembodiment, the thiadiazolin-3,5-dione compounds are useful in methodsfor preventing or treating neoplastic disease. In a further embodiment,the compounds are useful in methods for prevention or treating HPVinfection. In a further embodiment, the thiadiazolin-3,5-dione compoundsare useful in methods for preventing or treating HPV-E7 mediated E2Fdisplacement from pRb. In still another embodiment, thethiadiazolin-3,5-dione compounds are useful in methods for disruptingpRb/HPV-E7 complexes. In another embodiment, the thiadiazolin-3,5-dionecompounds are useful in methods for preventing or treating benignconditions caused by oncovirus containing an oncoprotein that targets,disrupts or inactivates pRb. In yet a further embodiment, thethiadiazolin-3,5-dione compounds are useful in methods for preventing ortreating genital warts. In another embodiment, thethiadiazolin-3,5-dione compounds are useful in methods for preventing ortreating neoplastic disease caused by human papilloma virus.

The term “neoplastic disease” as used herein refers to a disease orcondition in which a patient has an abnormal mass of tissue due to anabnormal proliferation of cells. The abnormal proliferation of cells mayresult in a localized lump, be present in the lymphatic system, or maybe systemic. In another embodiment, the neoplastic disease is caused byHPV infection, both low and high risk forms. In one embodiment, theneoplastic disease is benign. In another embodiment, the neoplasticdisease is pre-malignant, i.e., potentially malignant neoplasticdisease. In a further embodiment, the neoplastic disease is malignant,i.e., cancer.

The neoplastic diseases (cancers) caused by these oncoviruses arenumerous, but may be treated using the compounds, compositions andmethods described herein. In one embodiment, the neoplastic disease isan epithelial cancer, both low and high risk cancers. In anotherembodiment, the neoplastic disease is Kaposi's sarcoma (a skin cancerassociated with KSHV or HHV-8), Merkel cell carcinoma, hepatocellularcarcinoma (liver cancer), cervical cancer, anal cancer, penile cancer,vulvar cancer, vaginal cancer, neck cancer, head cancer, Kaposi'ssarcoma, multicentric Castleman's disease, primary effusion lymphoma,tropical spastic paraparesis, adult T-cell leukemia, Burkitt's lymphoma,Hodgkin's lymphoma, post-transplantation lymphoproliferative disease,nasopharyngeal carcinoma, pleural mesothelioma cancer of the lining ofthe lung), osteosarcoma (a bone cancer), ependymoma and choroid plexustumors of the brain, and non-Hodgkin's lymphoma. In another embodiment,the neoplastic disease is caused by HPV infection, both low and highrisk forms. In a further embodiment, the neoplastic disease is cervicalcancer. In a further embodiment, the neoplastic disease is anal cancer.In still a further embodiment, the neoplastic disease is penile cancer.In another embodiment, the neoplastic disease is vulvar cancer. In yet afurther embodiment, the neoplastic disease is vaginal cancer.

In another embodiment, the neoplastic disease is neck cancer. In still afurther embodiment, the neoplastic disease is head cancer such as eyecancer. Still other diseases caused by one of the exemplary pRbinactivating oncovirues are anticipated to be included with classicalneoplastic diseases as suitable for treatment with the compounds andmethods disclosed herein.

The term “benign” condition as used herein refers to a condition whichis not a neoplastic disease, i.e., the benign condition is not cancer.The benign condition is caused by an oncovirus containing an oncoproteinthat targets, disrupts or inactivates pRb, such as HPV. In oneembodiment, the benign condition is warts. In another embodiment, thebenign condition is skin warts such as common warts, plantar warts,subungal warts, or periungual warts, or flat warts. In a furtherembodiment, the benign condition is genital warts. In still anotherembodiment, the benign condition is anal warts. In yet a furtherembodiment, the benign condition is respiratory papillomatosis. Inanother embodiment, the benign condition is epidermodysplasiaverruciformis

I. The Thiadiazolidinedione Compounds

As discussed above, the inventors found thiadiazolin-3,5-dione compoundswhich are useful for treating neoplastic disease caused by oncogenicviruses. The term “thiadiazolin-3,5-dione compound” as used hereinrefers to compounds having the following backbone.

As noted by a

, the nitrogen atoms of this backbone are also bound to additionalsubstituents, thereby resulting in a stable chemical compound. In oneembodiment, at least one of the nitrogen atoms of this backbone is boundto a bulky substituent. “Bulky substituent” as used herein refers to achemical group that interferes with the ability of the viral oncoproteinto bind to the LxCxE motif of a pRb/E2F complex. In one embodiment, thebulky substituent is an optionally substituted aryl, optionallysubstituted heterocycle, optionally substituted heteroaryl, optionallysubstituted alkyl, or optionally substituted cycloalkyl. In anotherembodiment, the bulky substituent is an optionally substituted aryl. Ina further embodiment, the bulky substituent is an optionally substitutedphenyl.

In one embodiment, the thiadiazolin-3,5-dione compound has the structurenoted in formula (I):

In this structure, R¹ is selected from among optionally substitutedaryl, optionally substituted heteroaryl, and optionally substitutedheterocycle. In one embodiment. R¹ is

and R³ to R⁷ are, independently, selected from among H, optionallysubstituted alkyl, halogen, optionally and substituted alkoxy. Inanother embodiment, R¹ is

In a further embodiment, R¹ is

In still another embodiment, R¹ is any of these above-noted R³-R⁷containing structures and one of R³ to R⁷ is alkyl or alkoxy. In yet afurther embodiment, R¹ is any of these above-noted R³ or R⁷ containingstructures and R³ or R⁷ is alkyl. In another embodiment, R¹ is any oneof these above-noted R⁴ or R⁶ containing structures and R⁴ or R⁶ isalkyl. In still a further embodiment, R¹ is selected from among

R² in formula (I) is selected from among optionally substituted alkyl,optionally substituted cycloalkyl, and optionally substituted aryl. Inone embodiment, R² is C₁ to C₆ alkyl. In another embodiment, R² ismethyl, ethyl, or propyl. In a further embodiment, R² is optionallysubstituted aryl. In yet another embodiment. R² is

and R⁸ to R¹² are, independently, selected from among H, optionallysubstituted alkyl, halogen, and optionally substituted alkoxy. In afurther embodiment, R² is

and R⁸, R⁹, R¹¹, and R¹² are H and R¹⁰ is alkoxy. In still anotherembodiment, R² is

and R¹⁰ is OCH₃.

In another embodiment, the thiadiazolin-3,5-dione compound is:

An “alkyl” group as used herein refers to saturated aliphatichydrocarbon groups. An alkyl may have straight- or branched-chains. Inone embodiment, an alkyl group has 1 to about 10 carbon atoms (i.e., C₁,C₂, C₃, C₄, C₅ C₆, C₇, C₈, C₉, or C₁₀), or ranges there between. Inanother embodiment, an alkyl group has 4 to about 10 carbon atoms (i.e.,C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀), or ranges there between. In a furtherembodiment, an alkyl group has 5 to about 10 carbon atoms (i.e., C₅, C₆,C₇, C₈, C₉, or C₁₀), or ranges there between.

A “cycloalkyl” group as used herein refers to saturated aliphatichydrocarbon groups which are cyclic. In one embodiment, a cycloalkyl has3 to about 10 carbon atoms (i.e., C₃, C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀),or ranges there between. In another embodiment, a cycloalkyl has 5 toabout 10 carbon atoms (i.e., C₅, C₆, C₇, C₈, C₉, or C₁₀), or rangesthere between.

The terms “substituted alkyl” and “substituted cycloalkyl” refer toalkyl and cycloalkyl groups, respectively, having one or moresubstituents including, without limitation, hydrogen, halogen, CN, OH,NO₂, amino, aryl, heterocyclic, heteroaryl, alkoxy, and aryloxy.

“Alkoxy” refers to the group R—O— where R is an alkyl group, as definedabove. Exemplary C₁-C₆ alkoxy groups include but are not limited tomethoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy and t-butoxy.

The term “substituted alkoxy” refers to an alkoxy group having one ormore substituents on the alkyl chain including, without limitation,hydrogen, halogen, CN, OH, NO₂, amino, aryl, heterocyclic, heteroaryl,alkoxy, and aryloxy.

“Aryloxy” refers to the group R—O— where R is an aryl group, as definedbelow.

The term “substituted aryloxy” refers to an aryloxy group having one ormore substituents on the alkyl chain or aryl moiety including, withoutlimitation, hydrogen, halogen, CN, OH, NO₂, amino, aryl, heterocyclic,heteroaryl, alkoxy, and aryloxy.

The terms “substituted alkyl” and “substituted cycloalkyl” refer toalkyl and cycloalkyl groups, respectively, having one or moresubstituents including, without limitation, hydrogen, halogen, CN, OH,NO₂, amino, aryl, heterocyclic, heteroaryl, alkoxy, and aryloxy.

The term “halogen” as used herein refers to Cl, Br, F, or I groups.

The term “aryl” as used herein refers to an aromatic, carbocyclicsystem, e.g., of about 6, 7, 8, 9, 10, 11, 12, 13 to 14 carbon atoms,which can include a single ring or multiple aromatic rings fused orlinked together where at least one part of the fused or linked ringsforms the conjugated aromatic system. The aryl groups include, but arenot limited to, phenyl, naphthyl, biphenyl, anthryl, tetrahydronaphthyl,phenanthryl, indene, benzonaphthyl, and fluorenyl.

The term “substituted aryl” refers to an aryl group which is substitutedwith one or more substituents including halogen, CN, OH, NO₂, amino,alkyl, cycloalkyl, aryloxy, alkoxy, aryl, or heteroaryl. Desirably, asubstituted aryl group is substituted with 1, 2, 3, or 4 groups.

The term “heterocycle” or “heterocyclic” as used herein can be usedinterchangeably to refer to a stable, saturated or partially unsaturated3- to 9-membered monocyclic or multicyclic heterocyclic ring. Theheterocyclic ring has in its backbone carbon atoms and one or moreheteroatoms including nitrogen, oxygen, and sulfur atoms. In oneembodiment, the heterocyclic ring has 1 to about 4 heteroatoms in thebackbone of the ring. When the heterocyclic ring contains nitrogen orsulfur atoms in the backbone of the ring, the nitrogen or sulfur atomscan be oxidized. The term “heterocycle” or “heterocyclic” also refers tomulticyclic rings in which a heterocyclic ring is fused to an aryl ringof about 6, 7, 8, 9, 10, 11, 12, 13 to about 14 carbon atoms. Theheterocyclic ring can be attached to the aryl ring through a heteroatomor carbon atom provided the resultant heterocyclic ring structure ischemically stable. In one embodiment, the heterocyclic ring includesmulticyclic systems having 1, 2, 3, 4, or 5 rings.

A variety of heterocyclic groups are known in the art and include,without limitation, oxygen-containing rings, nitrogen-containing rings,sulfur-containing rings, mixed heteroatom-containing rings, fusedheteroatom containing rings, and combinations thereof. Examples ofheterocyclic groups include, without limitation, tetrahydrofuranyl,piperidinyl, 2-oxopiperidinyl, pyrrolidinyl, morpholinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, pyranyl, pyronyl, dioxinyl,piperazinyl, dithiolyl, oxathiolyl, dioxazolyl, oxathiazolyl, oxazinyl,oxathiazinyl, benzopyranyl, benzoxazinyl and xanthenyl.

The term “heteroaryl” as used herein refers to a stable, aromatic 5, 6,7, 8, 9, 10, 11, 12, 13 to 14-membered monocyclic or multicyclicheteroatom-containing ring. The heteroaryl ring has in its backbonecarbon atoms and one or more heteroatoms including nitrogen, oxygen, andsulfur atoms. In one embodiment, the heteroaryl ring contains 1 to about4 heteroatoms in the backbone of the ring. When the heteroaryl ringcontains nitrogen or sulfur atoms in the backbone of the ring, thenitrogen or sulfur atoms can be oxidized. The term “heteroaryl” alsorefers to multicyclic rings in which a heteroaryl ring is fused to anaryl ring. The heteroaryl ring can be attached to the aryl ring througha heteroatom or carbon atom provided the resultant heterocyclic ringstructure is chemically stable. In one embodiment, the heteroaryl ringincludes multicyclic systems having 1, 2, 3, 4 or 5 rings.

A variety of heteroaryl groups are known in the art and include, withoutlimitation, oxygen-containing rings, nitrogen-containing rings,sulfur-containing rings, mixed heteroatom-containing rings, fusedheteroatom containing rings, and combinations thereof. Examples ofheteroaryl groups include, without limitation, furyl, pyrrolyl,pyrazolyl, imidazolyl, triazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, azepinyl, thienyl, dithiolyl, oxathiolyl,oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl, oxepinyl, thiepinyl,diazepinyl, benzofuranyl, thionapthene, indolyl, benzazolyl, purindinyl,pyranopyrrolyl, isoindazolyl, indoxazinyl, benzoxazolyl, quinolinyl,isoquinolinyl, benzodiazonyl, napthylridinyl, benzothienyl,pyridopyridinyl, acridinyl, carbazolyl, and purinyl rings.

The term “substituted heterocycle” and “substituted heteroaryl” as usedherein refers to a heterocycle or heteroaryl group having one or moresubstituents including halogen, CN, OH, NO₂, amino, alkyl, cycloalkyl,aryloxy, alkoxy, aryl, or heteroaryl. A substituted heterocycle orheteroaryl group may have 1, 2, 3, or 4 substituents.

The compounds discussed above may encompass tautomeric forms of thestructures provided herein characterized by the bioactivity of the drawnstructures. Further, the compounds may also be used in the form of saltsderived from pharmaceutically or physiologically acceptable acids,bases, alkali metals and alkaline earth metals.

In one embodiment, pharmaceutically acceptable salts can be formed fromorganic and inorganic acids. Examples of useful organic and inorganicacids include, without limitation, acetic, propionic, lactic, citric,tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,camphorsulfonic, and similarly known acceptable acids.

In another embodiment, pharmaceutically acceptable salts may also beformed from organic and inorganic bases. Examples of useful inorganicbases include, without limitation, alkali metal salts such as, e.g.,sodium, lithium, or potassium, such as alkali metal hydroxides.Pharmaceutically acceptable salts may also be formed from organic bases,such as ammonium salts, mono-, di-, and trimethylammonium, mono-, di-and triethylammonium, mono-, di- and tripropylammonium (iso and normal),ethyldimethylammonium, benzyldimethylammonium, cyclohexylammonium,benzylammonium, dibenzylammonium, piperidinium, morpholinium,pyrrolidinium, piperazinium, 1-methylpiperidinium, 4-ethylmorpholinium,1-isopropylpyrrolidinium, 1,4-dimethylpiperazinium, 1-n-butylpiperidinium, 2-methylpiperidinium, 1-ethyl-2-methylpiperidinium, mono-,di- and triethanolammonium, ethyl diethanolammonium,n-butylmonoethanolammonium, tris(hydroxymethyl)methylammoniumphenylmonoethanolammonium, diethanolamine, ethylenediamine, and thelike. In one example, the base is selected from among sodium hydroxide,lithium hydroxide, potassium hydroxide, and mixtures thereof.

These salts, as well as other compounds, can be in the form of esters,carbamates, i.e., “pro-drugs”, which convert to the active moiety invivo. In one embodiment, the prodrugs are esters. In another embodiment,the prodrugs are carbamates. See, e.g., B. Testa and J. Caldwell,“Prodrugs Revisited: The “Ad Hoc” Approach as a Complement to LigandDesign”, Medicinal Research Reviews, 16(3):233-241, ed., John Wiley &Sons, 1996, which is herein incorporated by reference.

The compounds discussed herein also encompass “metabolites” which formby in vivo processing of the compounds.

II. Administration of the Thiadiazolin-3,5-Dione Compounds A.Compositions

The compounds of the invention may be formulated neat or with one ormore excipient for administration. One of skill in the art would readilybe able to determine suitable excipients based on the selectedthiadiazolin-3,5-dione compound, patient, administration route,disease/condition being treated, among others. Not only may thecomposition be solid or liquid, but excipient(s) may be solid and/orliquid carriers. The carriers may be in dry or liquid form and must bepharmaceutically acceptable. The compositions are typically sterilesolutions or suspensions.

Suitably, the thiadiazolin-3,5-dione compounds may be formulated fordelivery to a patient by any suitable route including, e.g.,transdermal, mucosal (intranasal, buccal, vaginal), oral, parenteral,intravenous, intratumoral, intranodal, among others. A variety ofsuitable delivery devices can be utilized for these delivery routes andinclude, without limitation, tablets, caplets, capsules, gel tabs,dispersible powders, granules, suspensions, injectable solutions,transdermal patches, topical creams or gels, and vaginal rings, amongothers.

In preparing the compositions described herein, thethiadiazolin-3,5-dione compounds may be combined with one or moreexcipients. Examples of excipients which may be combined with thethiadiazolin-3,5-dione compound include, without limitation, solidcarriers, liquid carriers, adjuvant, antioxidants, suspending agent,syrup, binders, buffers, coatings, coloring agents, compression aids,diluents, disintegrants, emulsifiers, emollients, encapsulatingmaterials, fillers, flavoring agents, glidants, granulating agents,lubricants, metal chelators, osmo-regulators, pH adjustors,preservatives, solubilizers, sorbents, stabilizers, sweeteners,surfactants, thickening agents, or viscosity regulators. See, theexcipients in “Handbook of Pharmaceutical Excipients”, 5^(th) Edition.Eds.: Rowe, Sheskey, and Owen, APhA Publications (Washington, D.C.),2005 and U.S. Pat. No. 7,078,053, which are incorporated herein byreference. The selection of the particular excipient is dependent on thenature of the thiadiazolin-3,5-dione compound selected and theparticular form of administration desired.

When the route of administration is oral, the composition may be anysuitable conventional form, including, without limitation, the form of acapsule, caplet, gel tab, dispersible powder, granule, suspension,liquid, thin film, chewable tablet, rapid dissolve tablet, medicallollipop, or fast melt. In one embodiment, the composition is a liquid.In a further embodiment, the composition is a solid. In anotherembodiment, the composition is a suspension. One of skill in the artwould readily be able to formulate the compositions discussed herein inany one of these forms.

Solid carriers include, without limitation, starch, lactose, dicalciumphosphate, microcrystalline cellulose, sucrose and kaolin.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringe ability exits. It must be stable underconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacterial and fungi. Thecarrier utilized in the injectable form may be a solvent or dispersionmedium containing, e.g., water, ethanol (e.g., glycerol, propyleneglycol and liquid polyethylene glycol), suitable mixtures thereof, andvegetable oil.

Liquid carriers may be utilized in preparing solutions, suspensions,emulsions, syrups and elixirs. In one embodiment, thethiadiazolin-3,5-dione compound is dissolved a liquid carrier. Inanother embodiment, the thiadiazolin-3,5-dione compound is suspended ina liquid carrier. In one embodiment, the liquid carrier includes,without limitation, water, e.g., sterile water, organic solvents (suchas glycerol, propylene glycol, liquid polyethylene glycol,dimethylsulfoxide (DMSO)), oils (such as fractionated coconut oil,arachis oil, corn oil, peanut oil, and sesame oil and oily esters suchas ethyl oleate and isopropyl myristate), fats, cellulose derivativessuch as sodium carboxymethyl cellulose, and non-ionic surfactants.

Adjuvants can include, without limitation, flavoring agents, coloringagents, preserving agents, and antioxidants, e.g., vitamin E, ascorbicacid, butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA).

In one embodiment, the thiadiazolin-3,5-dione compound may be combinedwith a suspending agent, including about 0.05, 0.1, 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 to about 5% of suspending agent.

In another embodiment, the thiadiazolin-3,5-dione compound may becombined with a syrup containing, e.g., about 10, 15, 20, 25, 30, 35,40, 45, to about 50% of sugar.

In a further embodiment, the thiadiazolin-3,5-dione compound may becombined with an elixir containing, e.g., about 20, 25, 30, 35, 40, 45to about 50% ethanol, and the like.

In another embodiment, the compositions may be utilized as inhalants oraerosols. When administered as an inhalant, the compositions may be influid unit doses using the thiadiazolin-3,5-dione compound and a vehiclefor delivery by an atomizing spray pump or by dry powder forinsufflation. When administered as an aerosol, the compositions may bein a pressurized aerosol container together with a gaseous or liquefiedpropellant, e.g., dichlorodifluoromethane, carbon dioxide, nitrogen,propane, and the like. Also optionally provided is the delivery of ametered dose in one or more actuations. When the compositions areadministered intranasally, the administration may be performed using amist or spray.

The thiadiazolin-3,5-dione compounds may also be administeredparenterally or intraperitoneally as solutions, suspensions,dispersions, or the like. Such pharmaceutical preparations may contain,e.g., about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, to about90%/6 of the thiadiazolin-3,5-dione compound in combination with thecarrier.

The thiadiazolin-3,5-dione compounds may also be administered via avaginal ring or transdermal patch.

The effective dosage or amount of the thiadiazolin-3,5-dione compoundsmay vary depending on the particular thiadiazolin-3,5-dione compoundemployed, the mode of administration and the severity of the conditionbeing treated. In one embodiment, the effective amount is about 0.01mg/kg to 10 mg/kg body weight. In another embodiment, the effectiveamount is less than about 5 g/kg, about 500 mg/kg, about 400 mg/kg,about 300 mg/kg, about 200 mg/kg, about 100 mg/kg, about 50 mg/kg, about25 mg/kg, about 10 mg/kg, about 1 mg/kg, about 0.5 mg/kg, about 0.25mg/kg, about 0.1 mg/kg, about 100 μg/kg, about 75 μg/kg, about 50 μg/kg,about 25 μg/kg, about 10 μg/kg, or about 1 μg/kg. However, the effectiveamount of the thiadiazolin-3,5-dione compound can be determined by theattending physician and depends on the condition treated, the compoundadministered, the route of delivery, the age, weight, severity of thepatient's symptoms and response pattern of the patient.

The effective amount of the thiadiazolin-3,5-dione compound may beprovided on regular schedule, i.e., daily, weekly, monthly, or yearlybasis or on an irregular schedule with varying administration days,weeks, months, etc. Alternatively, the effective amount to beadministered may vary. In one embodiment, the effective amount for thefirst dose is higher than the effective amount for one or more of thesubsequent doses. In another embodiment, the effective amount for thefirst dose is lower than the effective amount for one or more of thesubsequent doses. The number and frequency of dosages corresponding to acompleted course of therapy will be determined according to the judgmentof a health-care practitioner. The effective amounts described hereinrefer to total amounts administered for a given time period; that is, ifmore than one thiadiazolin-3,5-dione compound or a pharmaceuticallyacceptable salt thereof is administered, the effective amountscorrespond to the total amount administered.

These dosage regimens may be adjusted to provide the optimal therapeuticresponse. For example, several divided doses of each component may beadministered daily or the dose may be proportionally increased orreduced as indicated by the exigencies of the therapeutic situation. Inone embodiment, the compounds or compositions discussed herein may beadministered on a daily, monthly, or yearly basis. In one embodiment,daily administration is once. In another embodiment, dailyadministration includes divided units which are administered over thecourse of each day.

B. Additional Pharmaceutical Reagents

When utilized for these purposes, the thiadiazolin-3,5-dione compoundscan be administered in combination with other pharmaceutical agents, aswell as in combination with each other. The term “pharmaceutical” agentas used herein refers to a chemical compound which results in apharmacological effect in a patient.

The thiadiazolin-3,5-dione compounds described herein can beadministered to a patient in need thereof with one or more of thesepharmaceutical agents. In one embodiment, the thiadiazolin-3,5-dionecompounds are combined with one or more of these pharmaceutical agents,i.e., delivered to the patient concurrently. In another embodiment, thethiadiazolin-3,5-dione compounds are delivered to the patientconcurrently therewith one or more of these pharmaceutical I agents. Ina further embodiment, the thiadiazolin-3,5-dione compounds are deliveredprior to one or more of these pharmaceutical agents. In still anotherembodiment, the thiadiazolin-3,5-dione compounds are deliveredsubsequent to one or more of these pharmaceutical agents.

In one embodiment, the thiadiazolin-3,5-dione compounds may beadministered with a chemotherapeutic. One of skill in the art wouldreadily be able to select a chemotherapeutic for administration with thethiadiazolin-3,5-dione compounds based on the cancer being treated,patient, among others. In one embodiment, the chemotherapeutic isselected from among cisplatin, paclitaxel, topotecan, ifosfamide, or5-fluorouracil.

The thiadiazolin-3,5-dione compounds may also be administered with acompound which inhibits binding of HPV E6 to p53, i.e., “E6 inhibitor”.In one embodiment, the E6 inhibitor compound which inhibits binding ofHPV E6 to p53 is selected from among the following compounds. See,Baleja, “Identification of inhibitors to Paillomavirus type 16 E6protein based on three-dimensional structure of interacting proteins”,Antiviral Res., 72(1):49-59 (October, 2006) and D'Abramo and Archambault“Small molecule inhibitors of human papillovavirus protein-proteininteractions” Open Virol. J., 5: 80-95 (2011), which are hereinincorporated by reference.

The thiadiazolin-3,5-dione compounds may further be administeredconcurrently, subsequent, or prior to additional reagents which areutilized for immunotherapy and/or in vaccines. Desirably, theimmunotherapy and/or vaccines are tailored to the patient and specificdisease/conditions being treated. In one embodiment, the immunotherapyand/or vaccines are tailored to the patient and specific cancer beingtreated.

C. Additional Treatment Protocols

The thiadiazolin-3,5-dione compounds described herein may be utilized totreat patients afflicted with neoplastic disease by their administrationin conjunction with a non-chemical treatment protocol. For example,surgical debulking, in certain embodiments is a necessary procedure forthe removal of large tumor masses, and can be employed before, during orafter application of the methods and compositions as described herein.Chemotherapy and/or radiation therapy, in other embodiments, bolster theeffects of the therapy described herein. Finally, immune-based therapiescan eradicate residual disease and activate endogenous immune responses.Such combination approaches (surgery plus chemotherapy/radiation plusimmunotherapy) are anticipated to be successful in the treatment of manycancers along with the methods described herein.

Still other adjunctive therapies for use with the methods andcompositions described herein include, in one embodiment, acupuncture.In a further embodiment, the non-chemical treatment protocol is surgery.In yet another embodiment, the non-chemical treatment protocol ischiropractic care. In still another embodiment, the non-chemicaltreatment protocol is passive or active immunotherapy. In a furtherembodiment, the non-chemical treatment protocol includes X-rays. Instill another embodiment, the non-chemical treatment protocol includesultrasounds, among others. Still other method steps that can be includedwith or adjunctive to the methods described herein are diagnosticassessments, e.g., blood testing, to determine or monitor the progressof the infection, the course or status of the disease, relapse or anyneed for booster administrations of the compositions.

III. Kits Containing the Thiadiazolin-3,5-Dione Compounds

Also provided are kits or packages of pharmaceutical formulationscontaining (i) the thiadiazolin-3,5-dione compound discussed above andused herein; and (ii) a compound which inhibits binding of HPV E6 top53. In one embodiment, the thiadiazolin-3,5-dione compound is acompound of formula (I). Suitably, the kits contain one or morethiadiazolin-3,5-dione compounds as described herein and one or morecompound which inhibits binding of HPV E6 to p53. Advantageously, foruse in the kits, the thiadiazolin-3,5-dione compound and compound whichinhibits binding of HPV E6 to p53 are formulated for the desireddelivery vehicle and route. For example, the thiadiazolin-3,5-dionecompound and compound which inhibits binding of HPV E6 to p53 can beformulated for oral delivery, parenteral delivery, vaginal ring,transdermal delivery, or mucosal delivery as discussed in detail above.

In one embodiment, the kit is designed for delivery at home. The kitmay, therefore, include tubes or other containers, applicators, needles,syringes, and other appropriate packaging and instructions for use.

IV. Embodiments of the Methods

In one embodiment, a method for preventing disruption of pRb/E2Fcomplexes is provided and includes administering a compound of formula(I) to a patient in need thereof.

In another embodiment, a method for preventing disruption of pRb/E2Fcomplexes is provided and includes administering, to a patient in needthereof, a composition containing (i) a thiadiazolin-3,5-dione compoundcomprising an optionally substituted aryl group bound to one nitrogenatom of the thiadiazolin-3,5-dione compound; and (ii) a compound whichinhibits binding of HPV E6 to p53.

In a further embodiment, a method for preventing interaction between pRband a viral oncoprotein is provided and includes administering acompound of formula (I) to a patient in need thereof.

In yet another embodiment, a method for preventing interaction betweenpRb and a viral oncoprotein is provided and includes administering, to apatient in need thereof, a composition containing (i) athiadiazolin-3,5-dione compound comprising an optionally substitutedaryl group bound to one nitrogen atom of the thiadiazolin-3,5-dionecompound; and (ii) a compound which inhibits binding of HPV E6 to p53.

In still a further embodiment, a method for preventing or a diseasecaused by a virus carrying a viral oncoprotein containing a LxCxE motifis provided and includes administering a compound of formula (I) to apatient in need thereof.

In another embodiment, a method for preventing or a disease caused by avirus carrying a viral oncoprotein containing a LxCxE motif is providedand includes administering, to a patient in need thereof, a compositioncontaining (i) a thiadiazolin-3,5-dione compound comprising anoptionally substituted aryl group bound to one nitrogen atom of thethiadiazolin-3,5-dione compound; and (ii) a compound which inhibitsbinding of HPV E6 to p53.

In yet a further embodiment, a method for preventing or treatingneoplastic disease is provided and includes administering a compound offormula (I) to a patient in need thereof.

In still another embodiment, a method for preventing or treatingneoplastic disease is provided and includes administering, to a patientin need thereof, a composition containing (i) a thiadiazolin-3,5-dionecompound comprising an optionally substituted aryl group bound to onenitrogen atom of the thiadiazolin-3,5-dione compound; and (ii) acompound which inhibits binding of HPV E6 to p53.

In a further embodiment, a method for preventing HPV-E7 mediated E2Fdisplacement from pRb is provided and includes administering a compoundof formula (I) to a patient in need thereof.

In yet another embodiment, a method for preventing HPV-E7 mediated E2Fdisplacement from pRb is provided and includes administering acomposition containing (i) a thiadiazolin-3,5-dione compound comprisingan optionally substituted aryl group bound to one nitrogen atom of thethiadiazolin-3,5-dione compound; and (ii) a compound which inhibitsbinding of HPV E6 to p53.

In still a further embodiment, a method for disrupting pRb/HPV-E7complexes is provided and includes administering a compound of formula(I) to a patient in need thereof.

In another embodiment, a method for disrupting pRb/HPV-E7 complexes isprovided and includes administering a composition containing (i) athiadiazolin-3,5-dione compound comprising an optionally substitutedaryl group bound to one nitrogen atom of the thiadiazolin-3,5-dionecompound; and (ii) a compound which inhibits binding of HPV E6 to p53.

In yet a further embodiment, a method for preventing or treating genitalwarts is provided and includes administering a compound of formula (I)to a patient in need thereof.

In still another embodiment, a method for preventing or treating genitalwarts is provided and includes administering a composition containing(i) a thiadiazolin-3,5-dione compound comprising an optionallysubstituted aryl group bound to one nitrogen atom of thethiadiazolin-3,5-dione compound; and (ii) a compound which inhibitsbinding of HPV E6 to p53.

In a further embodiment, a method for preventing or treating neoplasticdisease caused by human papilloma virus, adenovirus, or SV40 is providedand includes administering a compound of formula (I) to a patient inneed thereof.

In yet another embodiment, a method for preventing or treatingneoplastic disease caused by human papilloma virus, adenovirus, or SV40is provided and includes administering a composition containing (i) athiadiazolin-3,5-dione compound comprising an optionally substitutedaryl group bound to one nitrogen atom of the thiadiazolin-3,5-dionecompound; and (ii) a compound which inhibits binding of HPV E6 to p53.

In any of the above methods, the selected compound may be formulated asdescribed above and administered via a route and in a dosage that isdeemed suitable by one of skill in the art, taking into considerationthe specific disease, the physical condition and status of the patientand any other relevant clinical symptoms.

The following examples are illustrative only and are not intended tolimit the present invention.

EXAMPLES Example 1 Analysis, Cultures, and Reagents (i) SpectroscopicAnalyses

Liquid Chromatography-Mass Spectral (LC-MS) analysis of the compoundsdiscussed in the examples was performed using a Waters Micromass® ZQ™system. The mobile phase contained 0.5% formic acid in H₂O andacetonitrile. The compounds were resolved on a Waters Sunfire™ C184.6×50 mm analytical column at a flow rate of 2.0 mL/min with a gradientof 10% to 90% acetonitrile over 6 minutes followed by 1 minute of 100%acetonitrile. Percent purity was calculated based on the UV absorptionchromatogram.

¹H-Nuclear Magnetic Resonance (NMR) analysis of the compounds discussedherein was performed on a Bruker AMX-500 spectrometer. Chemical shiftsare reported as δ values relative to internal chloroform (δ 7.27).

(ii) Cell Cultures

C-33A and SiHa cell lines (ATCC Nos. HTB-31™ and HTB-35™ ATCC celllines) and grown in 1× minimal eagle's media (MEM, Cellgro) supplementedwith fetal bovine serum (10%; Hyclone), penicillin-streptomycin (10μg/mL; Cellgro), L-glutamine (2 mM; Cellgro), sodium pyruvate (1 mM;Cellgro), and non-essential amino acids (100 μM; Gibco). HeLa and HCT116cell lines (ATCC Nos. CCL-2™ and CCL-247™ cell lines) were generousgifts from the laboratories of Susan Janicki, and Meenhard Herlyn,respectively, and maintained in the same way.

(iii) Expression and Purification of Proteins

The DNA encoding HPV16-E7_(CR2-3) (residues 17-98 of SEQ ID NO: 1 (NCBIsequence #2002324A)), HPV1A-E7_(CR2-3) (residues 16-93 of SEQ ID NO: 4(NCBI sequence # NP_040307)) and Ad5-E1A_(CR1-3) (residues 36-189 of SEQID NO: 2 (NCBI sequence # AP_000197)) were cloned into the pRSET vector,containing an N-terminal 6×-histidine tag. E. coli BL21(DE3) cells(Catalog No. 200131, Stratagene) transformed with these modified pPRSETvectors were grown to an OD₆₀₀ of 0.3 at 37° C. The temperature wasreduced to 25° C. for HPV-E7 expressing cells and to 18° C. for Ad5-E1Aexpressing cells, and the cells were induced with IPTG (1 mM) at anOD₆₀₀ of 0.5-0.7 and grown overnight. Following protein expression, thecells were centrifuged and frozen at −80° C. prior to purification.Cells were resuspended and lysed by sonication in a buffer containingTris (20 mM), pH=7.5, NaCl (500 mM), imidazole (35 mM), Zn(OAc)₂ (10μM), BME (10 mM) and 1×PMSF. The cell lysate was centrifuged at 18,000RPM and the resulting supernatant was loaded onto a Ni-NTA columnpre-equilibrated with Tris (20 mM), pH=7.5, NaCl (500 mM), imidazole (35mM), Zn(OAc)₂ (10 μM), and BME (10 mM). The column was washed and thebound protein was eluted using an imidazole gradient (35 mM to 250 mM).Fractions containing protein were concentrated and further purifiedusing size exclusion chromatography on a Superdex™ 200 analytical column(GE Healthcare Life Sciences) in a buffer containing Tris (20 mM),pH=7.5. NaCl (150 mM), and BME (10 mM).

For the use of pRb in the ELISA assay, DNA encoding pRb_(ABC) (residues376-928 of SEQ ID NO: 3 (NCBI sequence #P06400)) were cloned into thepFAST-Bac vector, containing an N-terminal GST tag. Protein wasexpressed in Sf9 cells for 48 hours before harvesting. The protein waspurified as described by the manufacturer (Novagen). The plasmidpGex6P-1-E2F1, encoding the marked-box and transactivation domain ofE2F1 (residues 243-437 of SEQ ID NO: 5 (NCBI Accession #NP_005216)) withan N-terminal GST tag, was provided by Dr. Steven Gamblin (MRC, MillHill, UK). GST-E2F1_(MB-TA) was expressed in E. coli BL21(DE3)CodonPlus® RIL cells (Novagen) for 5-6 hours at 30° C. and purified asdescribed in Liu et al., J Biol Chem 281:578-586 (2006). The GST tag wasremoved using PreScission® Protease reagent (GE Healthcare LifeSciences) as described in Liu et al., 2006 to yield an untaggedE2F1_(MB-TA) for assay purposes (Liu et al., 2006).

For use in pull-down studies, GST-tagged full-length HPV-E7 was clonedinto the pGEX-4T-1 vector, expressed in E. coli BL21(DE3) cells, andpurified as described by the manufacturer (Novagen). 6×His-pRb_(ABC)(residues 376-928 of SEQ ID NO: 3 (NCBI sequence #P06400)) was clonedinto the pRSET vector, expressed and purified as described above for the6×His-tagged proteins, except that Zn(OAc)₂ was excluded from thebuffers.

For use in isothermal titration calorimetry studies, untagged pRb_(AB)(372-787 with the linker from 590-635 removed) was prepared as describedin Xiao et al., PNAS USA, 100:2363-2368 (2003).

Example 2 Screening and Identification of pRb Antagonists

(i) Compound libraries

Approximately 88,000 compounds from several diverse small moleculelibraries were screened using the ELISA-based assay. Two thousandcompounds comprising the Spectrum Collection from MicroSource DiscoverySystems (Gaylordsville, Conn.) were tested at a final concentration of8.3 μM. A library of 14,400 chemically diverse compounds from MaybridgeHitFinder™ library (Cambridge, UK) was tested at a final concentrationof 12.5 μM. A third set of compounds, comprising 71,539 small molecules,from the orthogonally pooled screening (OPS) libraries, provided by theLankenau Chemical Genomics Center (Wynnewood, Pa.) were tested at afinal concentration (6.25 μM to 12.5 μM). The HitFinder™ and OPSlibraries were orthogonally compressed to contain 5 or 10 compounds perwell, respectively, and the data were deconvoluted based on methodssimilar to those described in Devlin et al., Drug Development Research,37(2):80-85 (February, 1996); Ferrand et al., Assay Drug Dev Technol3:413-424 (2005); and Motlekar et al., Assay Drug Dev Technol 6:395-405(2008).

The protein constructs employed were 6×His-HPV16-E7_(CR2-3) (residues17-98 of SEQ ID NO: 1 (NCBI sequence #2002324A)) harboring the LxCxEmotif of HPV-E7, GST-pRb_(ABC) (residues 376-928 of SEQ ID NO: 3 (NCBIsequence #P06400)) harboring the A/B pocket domain and C-terminal regionof pRb and untagged E2F_(MB-TA) (residues 243-437 of SEQ ID NO: 5 (NCBIAccession #NP_005216)) containing the marked-box and transactivationdomains of E2F that make pRb contact. 6×His-HPV16-E7_(CR2-3) wasmodified to improve its solubility and reduce its tendency to aggregateby substituting nonconserved cysteine residues with those found inlow-risk HPV1A-E7.

(ii) High Throughput Solution Screening and Data Processing

The assay employed an enzyme-linked immunosorbance assay (ELISA)performed as follows. In brief, the GST-pRb_(ABC)/E2F_(MB-TA) complexwas attached to a glutathione-coated 384-well microtiter plate and6×His-HPV16-E7_(CR2-3) in the presence of either 1% DMSO (negativecontrol) or 10 μM compound dissolved in DMSO. Inactive compounds had noeffect on HPV-E7 binding to pRb, which prevented formation of pRb/E2Fcomplexes. This resulted in unbound E2F, which was removed by anotherwash step. Compounds that inhibited HPV-E7-mediated disruption ofpRb/E2F complexes maintain E2F bound to the plate through pRb.Therefore, following plate washing, the amount of E2F remaining bound tothe plate was a measure of the potency of the compound in inhibitingHPV-E7-mediated disruption of pRb/E2F complexes. The amount of E2Fremaining bound to the plate was quantified by a bioassay using aprimary anti-E2F1 antibody. This was followed by a secondary antibodylinked to horseradish peroxidase that acts on the ELISA PicoChemiluminescent substrate, which was detected using anultrasensitive-luminometer detector.

The linear range of the assay was first determined by titrationexperiments measuring the amount of E2F remaining bound after incubationof the GST-pRb_(ABC)/E2F_(MB-TA) complex with serial dilutions of E7.

The screen was performed using automation in 384-well microtiter platesin screening buffer (20 mM Tris, pH=7.5, 150 mM NaCl and 0.05% Tween20).First, a complex was formed between 100 ng/100 μL GST-pRb_(ABC) and 10ng/100 μL E2F1_(MB-TA) that was incubated for 30-60 minutes. At the sametime, 20 μL of 500 nM 6×His-HPV16-E7_(CR2-3) was added to a 384-wellplate (Fisher Scientific) containing test compound (0.5 μL) dissolved inDMSO (or DMSO control) and allowed to incubate for 30-60 minutes. FortyμL of GST-pRb_(ABC)/E2F_(MB-TA) complex was then added to each wellcontaining 6×His-HPV16E7_(CR2-3) and test compound, and incubated atroom temperature for an additional 30-60 minutes. Fifty μL of theGSTpRb_(ABC)/E2F_(MB-TA)-16E7_(CR2-3) mixture was then transferred to apre-washed glutathione-coated 384-well plate (Thermo Scientific) andallowed to shake for 30 minutes. The plate was then washed with thescreening buffer and primary anti-E2F1 antibody (50 μL, Millipore)diluted 1:25,000 was added to each well and incubated for 60 minutes ona shaker. The plate was washed again and a goat anti-mouse IgGhorseradish peroxidase antibody (50 μL; BioRad) diluted 1:5,000 wasadded and incubated for 30 minutes on the shaker. After another set ofwashes, 50 μL of a 50:50 mixture of ELISA Pico ChemiluminescentSubstrate (Pierce) was added to each well and read within 20 minutesusing an Envision®s plate-reader (Perkin Elmer). The Janus® AutomatedWorkstation (Perkin Elmer) was used for liquid handling in an automatedHTS protocol.

Each plate receiving test compound also contained positive controls:GST-pRb_(ABC)/E2F_(MB-TA)+DMSO in columns 1 and 23 and negative controlsGST-pRb_(ABC)/E2F_(MB-TA)+16E7_(CR2-3)/DMSO in columns 2 and 24.Uniformity plates (192 positive controls, and 192 negative controls)were distributed throughout the screening plates to ensure both assayand result reliability. All compounds were screened in duplicate.

The Z′ factor parameter was used to assess the robustness of the assayduring automation in a 384-well format. The chemiluminescence signalfrom each well was normalized to the negative controls on each platebased on the following equation:

Z=(χ−μ)/σ

-   -   χ=chemiluminescence signal of a given well    -   μ=mean of the negative control population    -   σ=standard deviation of the negative control population

Generally, compounds giving a chemiluminescence signal higher than 3standard deviations above the mean were considered hits. Softwareapplications developed by CeuticalSoft (OpenHTS® depository) were usedto deconvolute the orthogonally compressed data for both the HitFinder®and OPS libraries. The data was grouped into four categories:

-   -   i. actives: compounds that displayed >50% inhibition of E2F        displacement and clearly mapped to a unique well in both the        horizontal and vertical directions)    -   ii. ambiguous: compounds that mapped to two or more wells in        either dimension    -   iii. orphan: compounds that displayed inhibition in only one        direction    -   iv. inactives compounds that displayed no inhibition

A three day replicate plate experiment consistently yielded Z′-factorsbetween 0.62 and 0.71 indicating that the assay was sufficiently robustfor valid drug screening (Zhang, 1999 cited above). This screenidentified 364 small molecule HPV-E7 inhibitors, yielding a primaryscreen hit rate of 0.41%, based on their effect of producing aluminescence signal greater than three standard deviations from the meanvalue. These compounds were selected and tested to confirm theiractivities and measure potency values. One hundred twenty of these 364compounds had IC₅₀ values of 15.6 μM or lower using the same assayformat as the primary screen, reducing the hit rate to 0.14%. Theremaining compounds either did not show reproducible inhibition, or werenot sufficiently potent to determine their IC₅₀ value and were discardedfrom further analyses.

The 120 confirmed “actives” were then tested in secondary assays asdescribed below to identify those with apparently selectivepharmacological activity in cells. A summary of the process for theidentification of confirmed screening hits is shown in FIG. 1.

(iii) Cytotoxicity in Cervical Cancer Cells

These 120 compounds were then analyzed for cytotoxicity in cervicalcancer cells either infected with HPV16 (SiHa) or negative for infectionwith HPV (C-33A) (Yee et al., 1985). The metabolic viability of cellswas measured using a MTS assay(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

Cultured cell lines were seeded in 384-well, clear, tissue cultureplates (NUNC) at 10.000 cells/well and 1,000 cells/well for C-33A andSiHa, respectively, and maintained overnight. These concentrations weredetermined based on each cell line's doubling time. The next day, thecompounds were independently dissolved in media (25 μM to 100 nM) to afinal DMSO concentration of 0.5%, were added to each well and incubatedwith cells for 48 hours. Cell viability was then monitored by additionof MTS reagent (8 μL; Promega) and measurement at A₄₉₀ using a WallacEnvision™ plate reader after 3 hours of incubation.

Staurosporine, which has the following structure and is a non-specifickinase inhibitor, was used as a positive control because it was expectedto be toxic in all cells (Ruegg and Burgess, 1989).

Out of the 120 compounds tested, 25 were selectively cytotoxic in SiHacells (HPV16) and not in C-33A (HPV negative) cells at concentrations ator below 6 μM. Of the 25 compounds that were selectively toxic in SiHacells, 7 had IC₅₀ values that ranged from 0.34 to 7.6 μM (FIG. 2A andTable 1).

The increase in apoptosis in SiHa cells upon treatment with thethiadiazolin-3,5-dione compounds illustrate that thethiadiazolin-3,5-dione compounds antagonize the ability of E7 to controlthe proliferation of the HPV-positive cell lines.

TABLE 2 Retention Purity Time Compound Structure (%) (min) 1

92.7 4.27 2

89.8 4.17 3

80.5 3.58 4

94.7 4.67 5

96.8 4.50 6

91.2 3.15 7

97.8 3.18(iv) Cytotoxicity in Cells Other than Cervical Cancer Cells

To eliminate the possibility that the seven thiadiazolin-3,5-dionecompounds identified above (Table 1) were cytotoxic to SiHa cells due totheir role in inactivating pRb, which is mutated in C-33A cells, theywere tested in additional cell lines TC-1, a mouse epithelial lineco-transformed with HPV 16 E6/E7 and c-Ha-Ras, HeLa, a human cell lineinfected with HPV 18 and HCT116, a human HPV negative colorectalcarcinoma cell line containing an intact retinoblastoma gene (DeFilippiset al., 2003; Scheffner et al., 1991; Yee et al., 1985).

Cultured cell lines were seeded in 384-well, clear, tissue cultureplates (NUNC) at 10,000 cells/well, 1,000 cells/well, 1,000 cells/well,1,000 cells/well, and 2,000 cells/well for C-33A, SiHa, HeLa, TC-1, orHCT116 cells, respectively, and maintained overnight. Theseconcentrations were determined based on each cell line's doubling time.The next day, compounds 1-9 independently dissolved in media to a finalDMSO concentration of 0.5%, were added to each well and incubated withcells for 48 hr. Cell viability was then monitored by addition of MTSreagent (8 μL; Promega) and measurement at A₄₉₀ using a Wallac Envision™plate reader after 3 hours of incubation.

As shown in FIG. 2B, the compounds were not cytotoxic in the HCT116 cellline, were cytotoxic in TC-1 cells, and were moderately cytotoxic inHeLa cells. Taken together, this data suggests that the seventhiadiazolin-3,5-dione compounds identified in the primary MTS areselectively cytotoxic in HPV infected cervical cancer cell lines.

Example 3 Inhibition of HPV-E7 Activity

Since HPV-E7 interacts with both pRb and E2F for disruption of thepRb/E2F complex, the ability of the seven compounds from Example 2 weretested for this ability to inhibit HPV-E7 activity by directlydisrupting HPV-E7 interactions with pRb (Liu et al., 2006).

(i) ELISA Assay

An ELISA assay was utilized with modifications such that the amount of6×His-HPV16-E7_(CR2-3) remaining bound to the partner protein on theplate could be measured. The compounds identified in Example 2 werepurchased (Lankenau Institute for Medical Research) as powders. Theirpurity was verified by LC/MS and their structures by NMR. IC₅₀ valueswere then measured for (i) the compounds identified in Example 1, (ii)the compounds prepared according to Examples 3-5, (iii)2-phenyl-4-methyl-1,2,4-thiadiazolidin-3,5-dione (available fromMolport, Latvia), and (iv)4-benzyl-2-methyl-1,2,4-thiadiazolidin-3,5-dione (TDZD-8; Catalog No.T8325-5MG, Sigma) using the same ELISA-based assay as described for thehigh-throughput screen, except that the assay was performed manually in96-well format and so all volumes used were double those from 384-wellformat.

All compounds were solubilized in DMSO (50 mM) and diluted for use inthe ELISA-based assay at a final DMSO concentration of less than 5%.Ten-fold dilutions of thiadiazolin-3,5-dione compound (starting at 100μM) were added to a mixture containing GST-pRb_(ABC) and6×His-HPV16E7_(CR2-3). The amount of E7 remaining was determined byadding a primary anti-His antibody. The concentrations of the compoundsin the IC₅₀ experiment spanned the range of enzyme activity from noinhibition to complete inhibition. Three independent IC₅₀ measurementswere performed for each compound and the average and standard deviationvalues are reported. All data was imported into the GraphPad® Software(Prism) for IC₅₀ determination. To calculate the IC₅₀ values, thedose-response curves were fit to one-site (Hill slope=1)sigmoidal-dose-response curves.

These data show that increasing the amount of compound led to adisplacement of 6×His-HPV16-E7_(CR2-3) from GST-pRb_(ABC), suggestingthe compounds prevent the interaction between these two proteins (FIG.3A).

(ii) Pull-Down Assays

To eliminate potential artifacts from this assay format, the ability ofthe thiadiazolin-3,5-dione compounds to disrupt HPV-E7/pRb interactionwas analyzed by performing pull-downs on Ni-NTA beads usingHis-pRb_(ABC) and GST-16E7_(FL).

Ten μg His-tagged protein pRb_(ABC) was incubated with 10 μL Ni-NTAbeads (Fisher) in a buffer containing Tris (20 mM), pH=7.5, NaCl (150mM), imidazole (35 mM) and Tween20® reagent (0.05%) for 15 minutes toallow the protein to bind. An equimolar amount of GST-HPV16-E7_(FL) wasthen added. Compounds 1-7, at various concentrations (0 μM, 0.01 μM, 0.1μM, 1 μM, 10 μM, and 100 μM), were independently added to each reactionmixture and allowed to incubate at 4° C. for one hour with gentleagitation. After one hour, each set of beads was spun at 500 g, unboundproteins were aspirated and the beads were washed with 1 mL bindingbuffer (20 mM Tris, pH=7.5, 150 mM NaCl, 35 mM Imidazole and 0.05%Tween20™ reagent). The beads were washed three times with this buffer,and then the beads were subjected to SDS-page analysis. The samples weretransferred to PVDF membrane to be visualized by western blotting.Anti-GST mouse monoclonal antibodies (1:2000) (Calbiochem) and anti-Hismouse monoclonal antibodies (1:5000) (Fisher) were used. Bands werevisualized by chemiluminescence (Pierce) and exposure to film (Kodak).See FIG. 3B.

As was shown using the ELISA method, the pull-down assay showed that anincrease in compound concentration lead to a displacement of GST-E7_(FL)from His-pRb_(ABC). The IC₅₀ values for the amount of respectivecompound required for 6×His-HPV16-E7_(CR2-3) displacement fromGST-pRb_(ABC), as determined by the ELISA assay, was within ten-fold ofthe corresponding IC₅₀ values of E2F displacement from GST-pRb_(ABC) inthe presence of 6×His-HPV16-E7_(CR2-3) (Table 2).

TABLE 2 IC₅₀ values for compounds that inhibit HPV-E7-mediateddisruption of pRb/E2F and disrupt pRb/viral oncoprotein complexes 16E71AE7 E1A (500 nM) (500 nM) (100 nM) K_(D) (μM) Compound pRb/E2F 7.6 ±1.2 10.6 ± 1.3  2.8 ± 2.2 1 pRb 11.2 ± 1.3  7.9 ± 2.1 5.0 ± 1.8 0.165 ±0.052 pRb/E2F 2.2 ± 1.6 3.5 ± 1.6 0.64 ± 2.3  2 pRb 0.57 ± 1.2  3.0 ±2.3 2.6 ± 1.3 0.104 ± 0.025 pRb/E2F 1.9 ± 1.3 4.5 ± 1.7 0.24 ± 2.0  3pRb 0.50 ± 1.5  3.4 ± 2.0 1.0 ± 2.1 0.106 ± 0.034 pRb/E2F 3.2 ± 1.3 5.5± 1.7 1.3 ± 2.2 4 pRb 4.5 ± 1.5 4.7 ± 2.1 3.8 ± 1.5 0.187 ± 0.022pRb/E2F 4.6 ± 1.3 5.5 ± 1.5 1.1 ± 2.5 5 pRb 3.2 ± 1.3 5.5 ± 2.7 3.5 ±1.7 0.210 ± 0.051 pRb/E2F 2.3 ± 1.6 3.3 ± 1.6 1.7 ± 2.7 6 pRb 0.40 ±1.4  1.3 ± 1.3 7.7 ± 1.7 0.381 ± 0.031 pRb/E2F 0.34 ± 1.9  3.5 ± 1.7 3.2± 2.7 7 pRb 0.29 ± 1.7  4.0 ± 2.5 2.8 ± 2.1 0.815 ± 0.070

Example 7 The Thiadiazolin-3,5-Dione Compounds Function by Binding topRb Through the LxCxE Binding Motif of Viral Oncoproteins

Since HPV-E7 mediates high affinity pRb binding through the associationof its LxCxE motif in CR2 to the B domain of pRb, the ability of thethiadiazolidinedione compounds to inhibit the ability of other LxCxEcontaining viral oncoproteins from disrupting E2F/pRb complexes wastested. The other LxCxE containing viral oncoproteins tested includedHPV-E7 from a low risk HPV form (type 1A) and Adenovirus E1A proteins.

A similar ELISA assay was used to assess the ability of the compounds toinhibit the ability of HPV16-E7-mediated disruption of E2F/pRbcomplexes. For these studies, 6×His-HPV1AE7_(CR2-3) and6×His-Ad5E1A_(CR1-3), containing the LxCxE motif, were employed. Theassay described above was modified in such a way that GST-pRb_(ABC)alone was added to HPV-E7_(CR2-3)+compound, HPV-E7_(CR2-3)+DMSO,Ad5-E1A_(CR1-3)+compound, or Ad5-E1A_(CR1-3)+DMSO. Mouse monoclonalanti-His antibody (Fisher) diluted 1:10,000 was used to detect how muchHis-E7_(CR2-3) remained bound to GST-pRb_(ABC) on the plate. Mousemonoclonal Ad5-E1A antibody (Abcam) diluted 1:10,000 was used to detecthow much E1A_(CR1-3) remained bound to GST-pRb_(ABC) on the plate. Allother steps remained unchanged.

To test the mode of inhibition by the thiadiazolin-3,5-dione compounds,each compound was first incubated with pRb for 30-60 minutes. Differentconcentrations of HPV-E7_(CR2-3), ranging from 50 μM down to 0.05 μM,were added to the GST-pRb_(ABC)+compound mixture and allowed to incubatefor 30-60 minutes. The reaction mixture was then transferred to aglutathione-coated plate, and shaken for 15-20 minutes. Mouse monoclonalanti-His antibody (Fisher) diluted 1:10,000 was used to detect how muchHPV-E7_(CR2-3) remained bound to GST-pRb_(ABC) on the plate.

As can be seen in FIG. 4A and Table 2, the compounds show similar levelsof inhibition as they did in the presence of 6×His-HPV16E7_(CR2-3).While the IC₅₀ values are lower for E1A, this is likely the result ofusing a lower concentration of E1A that showed linearity in the ELISAassay. The ability of the compounds to prevent an interaction betweeneither 6×His-HPV1AE7_(CR2-3) or 6×His-Ad5E1A_(CR1-3) with GST-pRb_(ABC)was also shown (FIG. 4B). The IC₅₀ values from these experiments rangedfrom 0.2-11.2 μM, which is comparable to the IC₅₀ values for compoundinhibition of HPV-16E7 mediated inhibition of E2F/pRb complexes (Table2). This data illustrate that the thiadiazolin-3,5-dione compoundsdisrupt the interaction between the pRb B domain and the LxCxE motif ofthe viral oncoproteins.

Example 8 Interaction of the Thiadiazolin-3,5-Dione Compounds with theStructured pRb B Domain

This example illustrates the ability of the thiadiazolin-3,5-dionecompounds to bind directly to a truncated pRb protein constructcontaining the A and B domains of the pRb pocket (pRb_(AB)) usingisothermal titration calorimetry (ITC).

Binding of compounds 1-9 to pRb_(AB) were measured by ITC using aMicroCal™ VP-ITC isothermal titration calorimeter (MicroCal, Inc).Proteins were extensively dialyzed against a buffer containing Hepes (20mM), pH=7.5. NaCl (150 mM) and Tris carboxy ethyl phosphine (0.1 mM)prior to analysis. Eight to twelve μL injections of 750-1500 μM compoundwere titrated into a pRb_(AB) solution (50-150 μM) pre-equilibrated to22° C. After subtraction of dilution heats, calorimetric data wereanalyzed with the MicroCal™ Origin® V5.0 (MicroCal Software,Northampton, Mass.).

The resulting integrated heat-flow spikes confirmed direct binding ofthiadiazolin-3,5-dione compounds to pRb with 1:1 stoichiometry andaffinities in the sub-micromolar range (FIG. 4C and FIG. 5A). A summaryof the dissociation constants is given in Table 2. To further confirmthat inhibitor binding was reversible, one of thepRb/thiadiazolin-3,5-dione compound complexes (pRb with compound 478166)was dialyzed overnight and ITC was repeated. As before, a binding curvewas obtained yielding a similar dissociation constant and stoichiometry,indicating that the thiadiazolin-3,5-dione compound was still able tointeract with pRb in a reversible fashion (FIG. 5B).

These results illustrate that the thiadiazolin-3,5-dione compounds binddirectly to pRb. These results also additionally suggest a route forstructure-based-drug design of additional HPV inhibitors.

Example 9 Determination of Concentration Thiadiazolin-3,5-Dione CompoundDependence

To determine if the thiadiazolin-3,5-dione compounds were competitivewith HPV-E7 for pRb binding or work through an allosteric mechanism, theELISA assay was utilized to measure the ability of HPV-E7 to displaceE2F from pRb as a function of thiadiazolin-3,5-dione compoundconcentration. As shown in FIG. 4D, the binding curves for6×His-HPV16-E7_(CR2-3) mediated displacement of E2F_(MB-TA) fromGST-pRb_(ABC) in the presence of varying concentrations of compound 3(0.025, 0.25, 0.5 and 5.0 μM), were obtained. The calculated K_(d),i.e., 140, 313, 304 and 764 nM, respectively, were above and below thedissociation constant of pRb (K_(d) for pRb of 104 nM). The bindingcurves showed a dependence on the concentration ofthiadiazolin-3,5-dione compound, where increasing thiadiazolin-3,5-dionecompound concentration is correlated with a rightward shift (higherapparent value) in the IC₅₀ values for HPV-E7 mediated displacement ofpRb/E2F complex.

These data illustrate that the thiadiazolin-3,5-dione compounds andHPV16-E7 bind competitively to pRb.

Example 10 Effect of the Thiadiazolin-3,5-Dione Compounds on Apoptosisin HPV-Infected Cells

Since the thiadiazolin-3,5-dione compounds bind to pRb, data wasgenerated regarding their effect in cells infected with HPV. To performthis example, SiHa cells (infected with HPV16) were employed since thethiadiazolin-3,5-dione compounds were most effective in this cell line.Cells were treated with either DMSO (at a final concentration of 0.5%)or 10 μM of thiadiazolin-3,5-dione compounds 3 and 4,2-(3,4-dimethyl-phenyl)-4-methyl-1,2,4-oxadiazolin-3,5-dione (which isan inactive oxo analog of compounds 1-7 and is available from LankenauInstitute for Medical Research), or 2 μM of staurosporine, which wasshown to be toxic using the MTS proliferation assay, for 48 hours. DNAcontent was determined by propidium iodine staining and analysis by flowcytometry.

Cultured cell lines were seeded in 60 mm tissue culture dishes (Falcon)at 1×10⁵ cells/well. The next day, compounds 1-9 (10 μM) or DMSO wereadded to each dish and allowed to incubate for 48 hours. Cells were thentrypsinized, washed with phosphate-buffered saline (1.0 mL; PBS), andfixed in ethanol (80%) for 30 minutes on ice. Fixed cells were spun at500 g for 5 minutes, rehydrated with PBS (1 mL), and spun again toremove any traces of ethanol. Cells were stained with propidium iodide(250 μL; PI), which was prepared by adding PI (100 μL, 2 mg/ml, Sigma)and RNase A (3.5 μL of 30 mg/ml, Sigma) into PBS (10 mL). Cells werethen analyzed at the Wistar Institute Flow Cytometry Core Facility usingstandard equipment, reagents, and methodologies known in the art.

The morphology of the cells was also noted. In agreement with ourbiochemical results and the MTS cell viability assay, compounds 3, 4,and staurosporine most drastically affected SiHa cells whereas theinactive analog had no effect (Table 3). DNA content analysis by flowcytometry indicated that the thiadiazolin-3,5-dione compounds caused anincrease of apoptotic SiHa cells as did the non-specific kinaseinhibitor staurosporine (Table 3). As noted, the percentage of cells inG0/G1 phase also decreased for these three compounds and the percentagesof apoptotic cells do not correspond well with the percent of viablecells as determined by the MTS assay, which may be due to the fact thatmost apoptotic cells float and are lost during collection for FACSanalysis. Another indicator of apoptosis was the fact that themorphology of SiHa cells treated with the thiadiazolidinedionesresembled that of those treated with staurosporine: they became rounderin shape and were predominantly floating in solution. Cells treated withDMSO and the inactive analog maintained the elongated shape inherent inSiHa cells. These results are consistent with the MTS data and in vitrodata, together supporting the interpretation that thethiadiazolin-3,5-dione compounds antagonize the proliferation ability ofHPV-E7.

TABLE 3 Comparison of Compounds 3 and 4, an inactive analog, andstaurosporine on the cell cycle and apoptosis in SiHa cells SiHa % G0/G1% G2/M % S % Apoptotic DMSO 72.9 15.9 10.7 1.1 Compound 3 57.7 21.1 15.46.5 Compound 4 49.2 20.0 15.8 15.2 Staurosporine 31.3 21.2 13.8 34.3Inactive analog 71.8 16.3 11.3 0.9

Example 11 Treatment of HPV in Mouse Model

Mice are generated as a model for human tumors associated with an HPVinfection, as described in Li, PNAS USA, 99(25): 16232-16236 (Dec. 10,2002). Prior to administration of test compound or control, each mousehas a palpable skin tumor that serves as a surrogate for a human tumorexpressing HPV-16 E7 protein, such as a human cervical carcinoma. Eachmouse is independently administered, s.c. or i.v., an effective amountof one of compounds 1-7 and a control.

Daily physical examinations of each mouse are conducted, eachexamination monitoring the presence and physical characteristics of theskin tumor. Additionally, blood samples are withdrawn from the micedaily over a period of 6 months and tested for viral loads of HPV, usingthe methods provided in the prior examples.

It is anticipated that compounds 1-7 reduce viral loads of HPV shortlyafter administration. It is also expected that no HPV remains in bloodsamples from the mice withdrawn at 6 months. It is further anticipatedthat compounds 1-7 result in the reduction in tumor size, eventuallyleading to total tumor loss.

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment is also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an”, refers to one or more, for example, “acompound” is understood to represent one or more compounds. As such, theterms “a” (or “an”). “one or more”, and “at least one” are usedinterchangeably herein. As used herein, the term “about” means avariability of 10% from the reference given, unless otherwise specified.Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application.

All publications and priority applications, including U.S. ProvisionalPatent Application No. 61/558,686, filed Nov. 11, 2011, now expired, andInternational Patent Application No. PCT/US2012/063683, filed Nov. 6,2012, now expired, and U.S. application Ser. No. 14/356,979 cited inthis specification are incorporated herein by reference. While theinvention has been described with reference to particular embodiments,it will be appreciated that modifications can be made without departingfrom the spirit of the invention. Such modifications are intended tofall within the scope of the appended claims.

What is claimed is:
 1. A composition for treating a human papillomavirus (HPV) mediated disease, said composition comprising (i) athiadiazolin-3,5-dione compound comprising an optionally substitutedaryl group bound to one nitrogen atom of said thiadiazolin-3,5-dionecompound; and (ii) a compound which inhibits binding of HPV E6 to p53.2. The composition according to claim 1, said thiadiazolin-3,5-dionecompound is of formula (I):

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 3. The composition according toclaim 2, wherein R¹ is:

wherein, R³ to R⁷ are, independently, selected from the group consistingof H, optionally substituted alkyl, halogen, optionally and substitutedalkoxy.
 4. The composition according to claim 3, wherein R¹ is:


5. The composition according to claim 4, wherein one of R³ to R⁷ isalkyl or alkoxy.
 6. The composition according to claim 2, wherein R² isC₁ to C₆ alkyl or of the structure:

wherein, R⁸ to R¹² are, independently, selected from the groupconsisting of H, optionally substituted alkyl, halogen, and optionallysubstituted alkoxy.
 7. The composition according to claim 6, wherein R⁸,R⁹, R¹¹, and R¹² are H and R¹⁰ is alkoxy.
 8. The composition accordingto claim 1, wherein said compound of formula (I) is:


9. The composition according to claim 1, wherein said compound whichinhibits binding of HPV E6 to p53 is selected from the group consistingof:


10. The composition according to claim 1, further comprising achemotherapeutic.
 11. A method for preventing disruption of pRb/E2Fcomplexes, said method comprising administering a compound of formula(I) or a composition of claim 1 to a patient in need thereof, whereinsaid compound of formula (I) is of the structure:

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 12. A method for preventinginteraction between pRb and a viral oncoprotein, said method comprisingadministering a compound of formula (I) or a composition of claim 1 to apatient in need thereof, wherein said compound of formula (I) is of thestructure:

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 13. A method for preventing ora disease caused by a virus carrying a viral oncoprotein containing aLxCxE motif, said method comprising administering a compound of formula(I) or a composition of claim 1 to a patient in need thereof, whereinsaid compound of formula (I) is of the structure:

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 14. The method according toclaim 13, wherein said viral oncoprotein is E1a from adenovirus, E7 fromHPV, or T-antigen from simian virus
 40. 15. A method for preventing ortreating neoplastic disease, said method comprising administering acompound of formula (I) or a composition of claim 1 to a patient in needthereof, wherein said compound of formula (I) is of the structure:

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 16. The method according toclaim 15, wherein said patient is infected with HPV or said neoplasticdisease is caused by HPV infection.
 17. A method for preventing HPV-E7mediated E2F displacement from pRb or disrupting pRb/HPV-E7 complexes,said method comprising administering a compound of formula (I) or acomposition of claim 1 to a patient in need thereof, wherein saidcompound of formula (I) is of the structure:

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 18. A method for preventing ortreating genital warts or neoplastic disease caused by human papillomavirus, adenovirus, or SV40, said method comprising administering acompound of formula (I) or a composition of claim 1 to a patient in needthereof, wherein said compound of formula (I) is of the structure:

wherein: R¹ is selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocycle; R² is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl, andoptionally substituted aryl; or a pharmaceutically acceptable salt,prodrug, solvate, or metabolite thereof.
 19. The method according toclaim 18, further comprising administering a chemotherapeutic.
 20. Themethod according to claim 18, further comprising treating said patientwith radiation.